Alternatively spliced variants of gamma-subunit of muscle-type acetylcholine receptor in fetal and adult skeletal muscle of mouse

Abstract

Gamma-subunit of nicotinic acetylcholine receptor is encoded by chrng gene of mouse. This gene is located on chromosome 1, spans 6.5 kb, and contains 12 exons and 11 introns. Previous studies have reported three transcript variants (C1-3) produced by alternative splicing; C1 contains all the 12 reported exons, C2 uses an in-frame alternate splice site in exon-2, and C3 produced by exon-5 skipping. These variants differ in their channel kinetics and opening times. In our study, we report the presence of two new transcript variants (T1 and T2) of chrng expressed in mouse postnatal day 3 and adult skeletal muscles. These transcripts contain novel first coding exon either N1 or N2. N1 is located in the 5' UTR, while N2 is an extended exon-2. 5' extension of exon-2 contains an initiation codon which produces a novel transcript variant. Either of the two exons can splice with the internal exons to produce mature transcripts making different 5' ends of the transcripts. Consequently, the proteins encoded by these two transcripts differ at N-termini. The presence of N2 exon containing transcript was further supported by the availability of EST from the database. These new variants display heterogeneous properties. They differ in the presence of signal peptide, phosphorylation, and acetylation of their amino acid residues of the new N-termini of the gamma subunit.

Fig. 1

Exon–intron organization of chrng gene and the transcript variants; exons and introns are presented by rectangles and interconnecting straight lines, respectively. Exons as part of transcript is shown with filled rectangles and empty rectangles represent the exon/part of exon skipped from the transcript. Splicing patterns are shown by bent lines between the exons. a Genomic DNA showing 12 exons and 11 introns in the gene. b Three different transcripts (C1, C2, and C3) that arise due to splicing and has been published earlier. C1 transcript contains all exons, C2 contains all exons but slightly shortened exon-2 (E2″), and C3 contains all exons except exon-5. c Newly identified two transcripts N1 and N2. Transcript N1 has a new 1st exon located upstream of the exon E1 that splices with exon-2 extended toward 3′ end. N2 transcript contains 5′ end extended exon-2 having internal initiation codon. Primer positions and directions are indicated by arrows above the exons. Exons and introns are not to scale. d Agarose gel electrophoresis of 5′ RACE product from P3 and adult skeletal muscle. Amplified 5′ RACE product was electrophoresed on 1.2 % gel and stained with ethidium bromide. M stands for 100 bp DNA ladder; +RT P3 +RT adult for RACE products in the presence of RT from P3 and adult skeletal muscle, respectively; and −RT P3 −RT adult are RACE products in absence of RT from P3 and adult skeletal muscle, respectively

Fig. 2

a Agarose gel electrophoresis of the RT-PCR products from P3 mouse muscle. 1 DNA marker 100 bp ladder. 2 RT-PCR products obtained using primers from the published sequence. It contains three bands each of which corresponded to the expected size of C1 (609 bp), C2 (546 bp), and C3 (453 bp). 3 Transcript variant T1 with anticipated size of 787 bp. 4 Transcript variant T2 with anticipated size of 674 bp. b Agarose gel electrophoresis of the RT-PCR products from adult mouse muscle. 1 DNA marker 100 bp ladder. 2 RT-PCR product for C3 with anticipated size of 453 bp. 3 Transcript variant T1 with anticipated size of 787 bp. 4 A very faint band of transcript variant T2 with anticipated size of 674 bp. c Multiple sequence alignment (ClustalW) of the deduced amino acid sequences of the published transcripts (C1, C2, and C3) and the newly identified transcripts (T1 and T2). Asterisks sign indicates the identical residues present in all five sequences. All sequences are identical from third exon onward. Accession numbers are given at the end of each sequence